J Parasit Dis (July-Sept 2019) 43(3):406–415 https://doi.org/10.1007/s12639-019-01105-7 ORIGINAL ARTICLE Antileishmanial activity and immune modulatory effects of benzoxonium chloride and its entrapped forms in niosome on Leishmania tropica 1 2 1 3 Maryam Hakimi Parizi • Abbas Pardakhty • Iraj sharifi • Saeedeh Farajzadeh • 4 5 1 Mohammad Hossein Daie Parizi • Hamid Sharifi • Ali Reza Keyhani • 1 6 1 Mahshid Mostafavi • Mehdi Bamorovat • Daryoush Ghaffari Received: 15 January 2019 / Accepted: 13 March 2019 / Published online: 29 March 2019 Ó The Author(s) 2019 Abstract Benzoxonium chloride is an anti-infective agent free solution and niosomal-encapsulated benzoxonium that is used as anti-septic drugs for disinfection of the chloride. Span/Tween 60 niosomal formulation of ben- mucus membrane, skin surface and anti-bacterial, and it is zoxonium chloride showed superior physical stability and also found to be effective against cutaneous leishmaniasis. high encapsulation efficiency (96%) than the other forms. The present study aims to evaluate the leishmanicidal Release from the formulations showed that the Span/ activity of benzoxonium chloride and niosomal forms Tween 60 containing drug had a milder gradient so that against Leishmania tropica stages. Benzoxonium chloride 10% of the drug was not released after 4 h. The benzox- niosomes were prepared by the thin film hydration method onium chloride and niosomal forms inhibited the in vitro and evaluated for morphology, particle size and release growth of promastigote and amastigote forms of L. tropica study and encapsulation efficiency. This study measured after 48 h of incubation and represented IC50 values of the cytotoxicity, leishmanicidal activity against pro- 90.7 ± 2.7 and 25.4 ± 0.6 lg/ mL, respectively. The rate mastigote and intra macrophage amastigote, apoptosis, and of apoptosis in niosomal formulations was approximately mRNA transcripts by quantitative real time PCR (qPCR) of equal to the positive control (meglumine antimoniate) at the same concentration. Also, an increase in the & Abbas Pardakhty 1 Leishmaniasis Research Center, Kerman University of [email protected]; [email protected] Medical Sciences, Kerman, Iran Maryam Hakimi Parizi 2 Pharmaceutics Research Center, Neuropharmacology [email protected] Institute, Kerman University of Medical Sciences, Iraj sharifi PO Box 76175-493, Kerman, Iran iraj.sharifi@yahoo.com 3 Department of Pediatric Dermatology, Kerman University of Saeedeh Farajzadeh Medical Sciences, Kerman, Iran [email protected] 4 Department of Pediatrics, Kerman University of Medical Mohammad Hossein Daie Parizi Sciences, Kerman, Iran [email protected] 5 HIV/STI Surveillance Research Center, WHO Collaborating Hamid Sharifi Center for HIV Surveillance, Institute for Futures Studies in sharifi[email protected] Health, Kerman University of Medical Sciences, Kerman, Iran Ali Reza Keyhani 6 [email protected] Research Center of Tropical and Infectious Diseases, Kerman University of Medical Sciences, Kerman, Iran Mahshid Mostafavi [email protected] Mehdi Bamorovat [email protected] Daryoush Ghaffari [email protected] 123 J Parasit Dis (July-Sept 2019) 43(3):406–415 407 concentration of this drug reduced the expression of IL-10, the mucus membrane, skin surface, and medical instru- but increased the expression of IL-12. The niosomal for- ments. Benzoxonium chloride is also effective against mulations provided improved anti-leishmanial activities of bacteria, certain protozoa, yeasts and non-spore forming benzoxonium chloride and played an immunomodulatory organisms (Kim et al. 2011). However, studies have sug- role as the mode of action in the treatment of anthroponotic gested that benzoxonium chloride inhibits certain bacterial CL. enzymes ([CSL STYLE ERROR: reference with no printed form.]). In 2015, Thio-Ben was tested in a clinical trial and Keywords Niosomal form Á Benzoxonium chloride Á it evaluated the efficacy of the topical use of tioxolone plus Leishmania tropica benzoxonium chloride (Thio-Ben) tincture in combination with cryotherapy as compared to intralesional meglumine antimoniate (glucantimeTM) along with cryotherapy in Introduction treating anthroponotic CL (ACL) (Daie Parizi et al. 2015). The study reported that the topical use of Thio-Ben com- Leishmaniasis refers to a disease that is caused by various bined with cryotherapy showed good efficacy in treating Leishmania species. It is a treatable and curable group of ACL along with benefits pertaining to more patient com- diseases that affects some of the poorest people on earth. pliance and less side effects than intralesional glucantime They are associated with malnutrition, population dis- (Daie Parizi et al. 2015). placement, poor housing conditions, and weak immune Recently, vesicular drug delivery systems such as system. Cutaneous leishmaniasis (CL), the most common phospholipid vesicles (liposomes) have been used in sev- form, causes skin lesions and it annually affects 0.7–1.2 eral types of leishmaniasis (Layegh et al. 2011; Balase- million people in the Americas, the Mediterranean basin, garam et al. 2012; Moosavian Kalat et al. 2014). Non-ionic the Middle East and Central Asia. In 2013, 95% of the surfactant vesicles (niosomes) have also been tested for the cases reported to WHO had occurred in 15 countries (Alvar treatment of leishmaniasis (Yasinzai et al. 2013). These are et al. 2012). In 2014, over 153,000 cases and 138,575 cases biodegradable, biocompatible, reduce cytotoxicity for ani- in 2015 (new cases and relapse cases) (World Health mal cells, more stable and relatively inexpensive, and they Organization (WHO) 2016) were reported to WHO from can be improved via the development of controlled and 10 high-burden countries—Afghanistan, Algeria, Colom- targeted drug-delivery formulations to overcome resistance bia, Brazil, Iran, Syria, Ethiopia, North Sudan, Costa Rica (Hu and Rhodes 1999). Given the above mentioned rea- and Peru (World Health Organization (WHO) 2016, 2018). sons, in this study, we investigated the in vitro effect of The clinical management of CL has been based only on benzoxonium chloride and its niosomal forms on Leish- treating patients with pentavalent antimonial compounds, mania tropica and also examined the cytotoxicity, apop- which are insufficient, toxic and cause resistance (Yasinzai tosis, and gene expression profiling of the selected et al. 2013). The mechanisms of classical drug resistance formulation. are often related with lower drug uptake, increased efflux, faster drug metabolism, drug target modifications, and the over-expression of drug transporters. The high prevalence Materials and methods of CL and the appearance of resistance to classical drugs highlight a demand for developing and exploring novel, Materials less toxic, low cost, and more promising therapeutic modalities (Yasinzai et al. 2013). Benzoxonium chloride was purchased from BOC Sciences, The topical treatment of CL presents an attractive USA. Meglumine antimoniate (MA, GlucantimeÒ) 1.5 g/ alternative that avoids the toxicities of parenteral therapy 5 mL solution was obtained from Sanofi-Aventis, France. while being administered through a simple painless route. MTT powder (3-[4, 5-dimethylthiazol-2-yl]-2, The treatments that have been used topically for leishma- niasis include paromomycin ointment, topical clotrimazole and miconazole, thermotherapy, cryosurgery using CO2 slush, cryotherapy by liquid nitrogen, CO2 laser, and cur- rently liposome of amphotericin B (Singh and Sivakumar 2004; Al-Qubati et al. 2012; Alavi-Naini et al. 2012; Mosimann et al. 2018). Benzoxonium chloride (C23H42•ClNO2, Fig. 1) belongs to quaternary ammonium compounds of anti-infective Fig. 1 Chemical structure of benzyl-dodecyl-bis (2-hydroxyethyl) agents that are used as anti-septic drugs and for disinfecting azanium;chloride (benzoxonium chloride) 123 408 J Parasit Dis (July-Sept 2019) 43(3):406–415 5-diphenyltetrazolium bromide; Thiazolyl blue), sorbitan release level was plotted against time to study the mech- esters (SpanÒ), their PEGylated derivatives (TweenÒ) and anism of drug release in order to compare the release cholesterol (CHOL) were provided by Sigma-AldrichÒ profiles of the different formulae. (USA). DMEM medium with sodium pyruvate, RPMI- For encapsulation efficiency level (EE%), non-en- 1640 medium with stable glutamine were purchased from trapped drug was separated from the niosome encapsulated Biosera (France) and fetal bovine serum (FBS) were pre- one by centrifugation at 14,000 rpm for 30 min. Three pared by Gibco (USA). Phycoerythrin (PE) Annexin V volumes, each of 1 mL isopropyl alcohol, were used to Apoptosis Detection Kit I (catalog number: 559763) was wash the isolated pellets, followed by re-centrifugation prepared by BD PharmingenTM. Phosphate buffered saline under the same conditions to completely remove the non- (PBS) was purchased from Cassion Lab (USA). Penicillin entrapped drug until a clear solution was obtained. After and streptomycin were obtained from Life Technology proper dilution, the benzoxonium chloride concentration (USA) and was frozen at - 20 °C until testing. was determined spectrophotometrically at 208 nm and analyzed by the following equation: % EE = (Total Preparation of niosomes drug - free drug/Total drug) 9 100. The thin film hydration method was used to prepare the In vitro assays niosomal forms (Pardakhty et al. 2012). Briefly, the non- ionic surfactants (Span 60 and 40 and Tween 60 and 40)/ The cytotoxicity effects of benzoxonium